A cultured rat mast cell line, the RBL-2H3 cell, was shown to produce the cytokine, TNFalpha, at physiologically active concentrations, in response to stimulation with antigen, or the combination of Ca2+-ionophore and phorbol myristate. Production and release of TNFalpha were two distinct processes although both were dependent on continued stimulation of the cell. Production of TNFalpha was dependent on Ca2+, partially dependent on activation of protein kinase C, and possibly on other pathways that utilized the G-protein Galphaz. Over-expression of Galphaz in transfected RBL-2H3 cells, for example, resulted in markedly enhanced production of TNFalpha and treatment with dexamethasone, which normally decreased expression of Galphaz and TNFalpha production, was less effective in reducing antigen-induced TNFalpha production in the transfected cells. There was no evidence that TNFalpha was incorporated into secretory granules, although the pathway for TNFalpha-release was totally dependent on protein kinase C. Inhibitors of protein kinase C blocked release of TNFalpha whereas such inhibitors only partially suppressed production of TNFalpha.